There are several mechanisms in place for the homeostatic regulation of biological processes, and balanced energy metabolism is critical to all of these mechanisms. In higher organisms energy stores are in the form of glycogen and upon energy deficit these stores are mobilized through enzymatic digestion to glucose-1-phosphate by the glycogen phosphorylase family of proteins.
Mammalian glycogen phosphorylases comprise a family of three isozymes which are distinguished by their electrophoretic mobilities, immunological properties and tissue-specific distribution. Each isozyme is encoded by a different gene, and these genes have been denoted PYGL, PYGM and PYGB, for liver, muscle and brain isoforms, respectively (Newgard et al., Crit. Rev. Biochem. Mol. Biol., 1989, 24, 69-99). The primary control however, common to all isozymes, is the phosphorylation of the inactive state, (b), to the active phosphorylated state, (a). This phosphorylation on serine-14 stabilizes the subunits of the homodimer and alters the binding sites for allosteric effectors and substrates (Sprang et al., Nature, 1988, 336, 215-221).
Liver glycogen phosphorylase (also known as 1,4-.alpha.-D-glucan:orthophosphate .alpha.-D-glucosyltransferase, glycogen phosphorylase (liver), EC 2.4.1.1 and HLGPa, for human liver glycogen phosphorylase a) is the enzyme which catalyzes the degradation of stored glycogen in the liver to glucose-1-phosphate by the cleavage of .alpha.-1,4-glycosidic bonds and therefore plays a critical role in carbohydrate metabolism and blood glucose homeostasis (Newgard et al., Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 8132-8136). The activity of liver glycogen phosphorylase is tightly regulated requiring the presence of a cofactor, pyridoxal phosphate, and involving allosteric mechanisms which include activation by AMP and glycogen binding and inhibition by glucose and glucose-6-phosphate binding. The enzyme is also regulated through phosphorylation by phosphorylase kinase which activates the homodimer (Keppens et al., Hepatology, 1993, 17, 610-614).
The gene for liver glycogen phosphorylase (PYGL) has been mapped to chromosome 14 and mutations in this gene give rise to glycogen storage disease type VI (GSD VI) or Hers Disease, a group of disorders that cause hepatomegaly and hypoglycemia (Burwinkel et al., Am. J. Hum. Genet., 1998, 62, 785-791; Chang et al., Hum. Mol. Genet., 1998, 7, 865-870; Newgard et al., Am. J. Hum. Genet., 1987, 40, 351-364). These mutations consist of two splice-site mutations which result in aberrant exon retention and exon skipping and two missense mutations which produce nonconservative replacements of amino acids that are normally conserved in all eukaryotes.
To date, two types of inhibitors targeting glycogen phosphorylase function have been reported. These involved the use of glucose analogs containing multiple polar groups which bind near the active site of the protein (Lundgren et al., 1997; Lundgren and Kirk, 1995) and caffeine and other heteroaromatic analogs which bind at the purine inhibitory site (Kasvinsky et al., Can. J. Biochem., 1981, 59, 387-395; Kasvinsky et al., J. Biol. Chem., 1978, 253, 3343-3351; Kasvinsky et al., J. Biol. Chem., 1978, 253, 9102-9106). However, none of these compounds have been shown to be orally active, limiting their utility.
Recently, the discovery of an orally active compound, CP-91149 and derivatives thereof, that lowers plasma glucose levels in an animal model of type 2 diabetes was reported (Hoover et al., J. Med. Chem., 1998, 41, 2934-2938; Martin et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1776-1781). This indole-containing compound was shown to inhibit glycogenolysis in diabetic ob/ob mice, and in rat and human liver cells by inhibiting liver liver glycogen phosphorylase. It is believed that inhibition of glycogenolysis will be of therapeutic benefit in the treatment of diabetes, particularly type II diabetes.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of glycogen phosphorylase expression.